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BMC Evolutionary Biology 2008, 8:56                            http://www.biomedcentral.com/1471-2148/8/56




            was HKY+G (AIC; 10 M generations; burnin = 1500, the  stitutional time (τ = 2µt; t is the time in generations, µ the
            same settings were used for the 16S rRNA sequences). For  substitution  rate per locus and  per generation [89]).
            RAG-1 fragments (860 bp, model: GTR+G; AIC, burnin =  Graphically, the mismatch distribution of a recently
            1000) and  the  alpha-tropomyosine intron  (612  bp;  expanded population is unimodal and smooth; the wave-
            model: HKY+G; AIC; because of the occurrence of inser-  shaped curve is centered nearer the y-axis the more recent
            tions and deletions, all missing and ambiguous data were  the expansion, moving away as the number of mis-
            excluded and only 405  bp  were  analyzed; 5 M genera-  matches increases [89]. By letting θ become very large, θ 0
                                                                                             1
            tions; burnin = 1000).                              and  τ are estimated from the data [90]. We set  θ to
                                                                                                             1
                                                                1,000,000. We assessed the deviation of the observed dis-
            Because amplification or alignment of markers was not  tribution  from the expected under a model of sudden
            equally possible for identical  outgroup taxa  from the  expansion by comparing the raggedness statistics of the
            available material, more than one species had to serve as  observed distribution with a simulated distribution  to
            outgroup. In  previous analyses [[28] and unpublished  determine the probability  that the raggedness  of the
            data], we demonstrated that all used taxa (Bufo surdus, B.  observed distribution could have arisen by chance. Third,
            raddei, B.  calamita, B.  bufo) represent suitable outgroup  we estimated Tajima's D [91] in DnaSP for each grouping.
            species.                                            Tajima's test of selective neutrality compares two θ estima-
                                                                tors and its significance is evaluated by comparison of the
            Taxonomic subdivision                               test statistic (D) with values randomly generated under
            To evaluate the distinctiveness of genetic groups of West-  "neutrality." Significant values indicate the population
            Mediterranean green toads  of  the  B. viridis  subgroup  has deviated from neutrality, or that another demographic
            [sensu [28]], and of further geographic subdivisions  force has caused the deviation from expectation, such as
            within each genetic group, we calculated pairwise F val-  population expansion (significant negative D).
                                                       ST
            ues between all groups using Arlequin 2.0 [85] based on
            the mitochondrial control region.                   Divergence times among the main mitochondrial lineages
                                                                were estimated using a Bayesian-coalescence approach, as
            Demographic analyses and divergence time estimates  implemented in BEAST 1.4.6 [92-95]. In analysis of the
            We assessed the possibility of population expansion for  control region, we used a matrix of 60 individuals and 752
            green toad groupings assembled into different geographi-  bp. We started the search with an UPGMA tree, constrain-
            cal populations. We applied three  different  tests,  each  ing the clade  B. boulengeri-B. siculus  from Sicily to be
            with different strengths, to the mitochondrial  control  monophyletic. Because we were analyzing a species-level
            region dataset (846 or 541  bp), to detect evidence of  phylogeny, we used a Yule tree prior, which assumes a
            recent expansion. First, we applied Fluctuate [86], a maxi-  constant speciation rate per lineage. We applied an uncor-
            mum-likelihood estimator of the parameters θ and g (θ =  related relaxed molecular clock, with the substitution rate
            2Ne/µ; g = exponential population growth rate parame-  of the branch lengths being sampled from a prior normal
            ter). The exponential growth parameter (g) was used to  distribution with a mean value of 0.02 and a standard
            estimate the size of the population at time in the past from  deviation of 0.007 [94]. The search was conducted for a
            N = θ e-(gµ)t  where N is the effective population size at time  range of substitution rates that varied from 1% to 3% per
              t
                            t
            t in the past [86]. Using this equation, t was estimated by  million years. We ran four independent analyses for 20 ×
                                                                  6
            substituting N with N /N t = 0  = 0.1 (µ is DNA substitution  10 generations. We checked for convergence and station-
                               t
                        t
            rate per site per generation, N is the female effective pop-  arity of the different analyses in Tracer 1.4 and combined
                                     t
            ulation size at time t). Repeated analyses to ensure stabil-  the results in the BEAST module LogCombiner 1.4.4 (after
            ity of estimates were run as described by Stöck et al. [28].  removing the first 2 × 10 generations from each analysis
                                                                                     6
            Growth was inferred  using logarithmic likelihood ratio  as "burnin").
            tests with one degree of freedom [87].
                                                                In analysis of the 16S rRNA, we modified the search strat-
            Second, DnaSP version 4.0 [88] was used to calculate and  egy to overcome the slow rate of convergence and station-
            show in graphic form the distributions of observed and  arity of the MCMC chains. We analyzed a matrix of 80
            expected pairwise nucleotide site differences, also called  individuals and 512 bp. We first constructed a UPGMA
            mismatch  distributions, between all individuals  within  tree with maximum likelihood distances (model selected
            each group, and the respective expected values for grow-  in Mr.ModelTest v.2: TrN + I), which was specified as the
            ing populations [89]. The model of sudden expansion  starting tree in Beast. Previous studies have used values of
            describes an initial population at equilibrium, with the  0.33% for the 16S rRNA or 0.7% more generally for a vari-
            expected pairwise differences, θ , (θ = 2N µ) and assumes  ety of different regions of the mitochondrial genome [e.g.,
                                      0
                                               e
            rapid population growth, resulting in  θ (Theta final).  [78,79]]. We specified the prior for the mean substitution
                                               1
            Tau, τ, is the time of the growth measured in units of sub-  rate as a normal distribution, with a mean of 0.008 and
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